A major scientific challenge is to understand the molecular events that drive the evolution of premalignant lesions in actual tissue. Laser capture microdissection (LCM) was originated to provide a reliable method to procure pure populations of cells from specific microscopic regions of tissue sections; in one step, under direct visualization. The cells of interest are transferred to a polymer film that is activated by laser pulses. The exact morphology of the procured cells (with intact DNA, RNA and proteins) is retained and held on the transfer film. LCM technology has been successfully applied to DNA, and RNA analysis from frozen and fixed embedded tissue. In the past it has not been possible to extract, quantify and characterize the functional state of specific proteins expressed by individual subpopulations of cells in actual tissue. Consequently, an important ongoing and future goal is to extend our microdissection technology to include the molecular profiling of cancer progression into the realm of quantitative proteomics: characterization of known proteins, as well as discovery of new proteins associated with progression. We have developed reverse phase protein microarrays and have used them to correlate the state of signal pathways associated with response to experimental therapy. The ultimate goal is to individualize therapy based on a tumor molecular profile of the signal network.In recent studies these techniques have been applied to follicular lymphoma, to develop prognostic profiles, and better map molecular pathways of disease. Follicular lymphoma (FL) is the second most common non-Hodgkin's lymphoma and generally is incurable. Reliable prognostic markers to differentiate patients who progress rapidly from those who survive for years with indolent disease have not been established. Most cases overexpress Bcl-2, but the pathogenesis of FL remains incompletely understood. To determine whether a proteomic approach could help overcome these obstacles, we procured lymphoid follicles from 20 cases of FL and 15 cases of benign follicular hyperplasia (FH) using laser capture microdissection. Lysates were spotted on reverse-phase protein microarrays and probed with 21 antibodies to proteins in the intrinsic apoptotic pathway, including those specific for posttranslational modifications such as phosphorylation. A panel of three antibodies [phospho-Akt(Ser473), Bcl-2, and cleaved poly(ADP-ribose) polymerase] segregated most cases of FL from FH. Phospho-Akt(Ser473) and Bcl-2 were significantly increased in FL (P = 0.001 and P < 0.0001, respectively). Additionally, the Bcl-2/Bak ratio completely segregated FL from FH. High ratios of Bcl-2/Bak and Bcl-2/Bax were associated with early death from disease with differences in median survival times of 7.3 years (P = 0.0085) and 3.8 years (P = 0.018), respectively. Using protein microarrays, we identified candidate proteins that may signify clinically relevant molecular events in FL. This approach showed significant changes at the posttranslational level, including Akt phosphorylation, and suggested new prognostic markers, including the Bcl-2/Bak and Bcl-2/Bax ratios. Proteomic end points should be incorporated in larger, multicenter trials to validate the clinical utility of these protein microarray findings.